Polypeptide competitor for immunoglobulin E

ABSTRACT

A competitor for human Immunoglobulin E (IgE) comprises a polypeptide which has a core sequence of seventy-six amino acids which is shown, together with the corresponding DNA sequence coding therefor, in FIG. 2. This amino acid sequence, numbered 1 to 76, corresponds to amino acids 301 to 376 of the epsilon heavy cain of IgE. The polypeptide may also include additional short sequences at the beginning and/or end of the core sequence which are physiologically harmless and do not contribute to the ability of the core sequence to bind compete with native IgE for the high-affinity receptor sites on human cells. The polypeptide is indicated for the treatment of Type I hypersensitivity reactions such as hay fever. The polypeptide may be produced synthetically or by expression from Escherichia coli containing a plasmid having a DNA segment coding for the polypeptide.

This is a continuation of application Ser. No. 07/295,033 filed Feb. 24,1989, now abandoned.

This invention relates to a polypeptide competitor for humanImmunoglobulin E (IgE). More particularly the invention relates to apolypeptide which is capable of binding specifically to the highaffinity Fc receptor sites for IgE which exist on human cells,particularly on mast cells and basophils, thereby inhibiting thebiological responses, such as exocytosis or degranulation, which takeplace when antigen specific IgE binds to and crosslinks such receptorsites in the presence of antigen. The invention also relates topharmaceutical preparations in which the polypeptide is an activeconstituent. The invention further relates to a method for thepreparation of the polypeptide using genetically modified bacteria.

In the human immune system, the principal role of IgE is believed to beto provide immunity to parasites. It also, however, mediates Type Ihypersensitivity which is an allergic response leading to themanifestation of such symptoms as hay fever and asthma. Briefly, themechanism of the allergic response is that on encountering a normallyinnocuous antigen such as pollen, synthesis of antigen-specific IgE byB-cells is initiated. The antigen-specific IgE then binds to mast cellreceptor sites via its Fc region and thereafter any further encounterwith the antigen triggers degranulation of the mast cells releasingmediators, principally histamine, resulting in the acute inflammatorysymptoms typical of Type I hypersensitivity.

Structurally, IgE, in common with the other immunoglobulins, comprisestwo heavy and two light chains, the epsilon heavy chain having fivedomains, a variable domain VH and constant domains CH1 to CH4. Themolecular weight of IgE is in the region of 188,000 of which the heavychain accounts for about 72,500, representing a sequence ofapproximately 550 amino acid residues.

It has been reported (Nature, vol.315, 1985, No.6020, pp 577-578) that apeptide sequence of 330 amino-acids corresponding to amino acid residues218 to 547 (in accordance with the numbering given by Bennich, Progressin Immunology II, Vol I, July 1974, pp 49-58) of the epsilon heavy chainof IgE has an inhibitory effect on the release of mediators from humanmast cells. The numbering is erroneously assigned in that paper inNature; the more correct numbering would be 208 to 537. The 330amino-acid sequence exists as a dimer consisting of two chains ofamino-acids, each of 330 amino-acids in length, linked by disulphidebonds.

U.S. Pat. Nos. 4,171,299 and 4,161,522 disclose that an oligopeptidecontaining from three to ten amino acids in a sequence selected from aportion of amino acids 265 to 537 of the Bennich nomenclature (seereference above) of the Fc region of human IgE will block Fc receptorsof mast cells thus inhibiting degranulation and release of mediatorssuch as histamine. The most active of these oligopeptides is identifiedas the pentapeptide Asp-Ser-Asp-Pro-Arg (called HEPP: HumanImmunoglobulin E Polypeptide) derived from the amino acid sequence 320to 324 of the IgE heavy chain. In native IgE amino acid 322 isasparagine, but it is suggested in the Patents that substitution ofasparagine by aspartic acid leads to a substantial enhancement of theblocking activity.

In the Patents mentioned above the full sequence which is attributed toBennich (Progress in Immunology II, Vol I, July 1974, pp 49-58) isquoted and shows aspartic acid at location 322. However, Bennich himselflater asserts (Int. Arch. Allergy Appl. Immunol. 53, 459) thatasparagine resides at that location. Bennich also reports that neitherof the peptides Asp-Ser-Asp-Pro-Arg nor Asp-Ser-Asn-Pro-Arg has anyblocking activity. Determination of the gene sequence has shown thatamino acid 322 is asparagine and not aspartic acid. In European PatentApplication No. 102634 asparagine and not aspartic acid is correctlyquoted at the equivalent location.

Further, it is also reported that the specific activity of HEPP is lowrequiring excessively large doses for any significant physiologicaleffect.

It is knwon that IgE epsilon chain fragments may be synthesised inEscherichia coli by cloning and expression of the DNA sequences codingfor the appropriate domains of the IgE chain (Eur. J. Immunol. 1985, 15:966-969 and Proc. Natl. Acad. Sc. USA, vol.81, 1984, 2955-2959).

An object of the present invention is to provide a new peptide for usein anti-allergy treatment.

According to the present invention there is provided a polypeptidecompetitor for human Immunoglobulin E (IgE), comprising a monomericchain of amino acids having the having following sequence:

Gln-Lys-His-Trp-Leu-Ser-Asp-Arg-Thr-Tyr-

Thr-Cys-Gln-Val-Thr-Tyr-Gln-Gly-His-Thr-

Phe-Glu-Asp-Ser-Thr-Lys-Lys-Cys-Ala-Asp-

Ser-Asn-Pro-Arg-Gly-Val-Ser-Ala-Tyr-Leu-

Ser-Arg-Pro-Ser-Pro-Phe-Asp-Leu-Phe-Ile-

Arg-Lys-Ser-Pro-Thr-Ile-Thr-Cys-Leu-Val-

Val-Asp-Leu-Ala-Pro-Ser-Lys-Gly-Thr-Val-

Asn-Leu-Thr-Trp-Ser-Arg

The core sequence of seventy-six amino acids defined above is capable ofbinding to human IgE high-affinity receptor sites. The said sequence,numbered in FIG. 2 as 1 to 76, corresponds to the amino acid sequencespanning residues 291 to 366 (Bennich nomenclature) of the heavy chainof human IgE.

In addition the core sequence may have short sequences of amino acidsinitiating (X--) and terminating (--Y) the core sequence and covalentlyattached to its 3' and/or 5' ends, which do not participate in thebinding to IgE receptors and which are not physiologically harmful.

Further according to the invention there is provided a DNA having thefollowing nucleotide sequence:

CAG AAG CAC TGG CTG TCA GAC CGC ACC TAC

ACC TGC CAG GTC ACC TAT CAA GGT CAC ACC

TTT GAG GAC AGC ACC AAG AAG TGT GCA GAT

TCC AAC CCG AGA GGG GTC AGC GCC TAC CTA

AGC CGG CCC AGC CCG TTC GAC CTG TTC ATC

CGC AAG TCG CCC ACG ATC ACC TGT CTG GTC

GTC GAC CTG GCA CCC ACC AAG GGG ACC GTG

AAC CTG ACC TGG TCC CGG.

The present invention also provides a transformant in which the DNAcontains the nucleotide sequence defined above. Preferably thetransformant host is Escherichia coli

Most preferably the transformant comprises Escherichia coli N4830harbouring the plasmid pE 2-3 (Accession Number NCTC 11993, depositedwith the National Collection of Type Cultures, London on Jul. 1st.1986).

The invention further provides a vector in which the DNA is as definedabove, said segment being oriented within said vector such that in ahost said segment is expressed to produce a polypeptide. The inventionalso provides a host organism transformed by the aforesaid vector.

Further according to the present invention there is provided a method ofpreparing the polypeptide defined above, comprising culturing theaforesaid host organism and isolating the polypeptide from the culture.

Using this method the resultant polypeptide product may include thegroups X and Y defined above. If thought necessary or desirable thesegroups may be removed from the core sequence of amino acids by standarddegradation procedures but, being physiologically harmless, there is nocompelling reason for removing them. In the specific example which willbe given later, the group represented by X is NH₂ -Met-Asp- and thegroup represented by Y is -Leu-Ile-Asn.

Alternatively the polypeptide may be synthesised by known chemicalsynthetic methods.

This invention also includes a pharmaceutical preparation in which theactive principle is the polypeptide defined above.

The preparation may also include a pharmaceutical carrier permittingadministration of the polypeptide in an appropriate manner, for exampleintranasally.

The polypeptide of the invention may also be covalently linked to orassociated with other therapeutic or diagnositic agents or othermolecules with the effect that the polypeptide acts to target thetherapeutic or diagnostic agent to cells bearing IgE high-affinityreceptors.

By way of explanation, the core sequence of the polypeptide of thisinvention bridges the second and third domains of the epsilon chainconstant region of IgE. Previous work (J. Immunol 114, 1838, 1975;Immunol. Rev. 41, 3, 1978) concluded that both the second and fourthdomains were required to form the binding site. It was thereforeunexpected that in respect of polypeptides with a major deletion in theCH2 domain, a major deletion in the CH3 domain and deletion of theentire CH4 domain or a combination of these deletions a binding abilitycomparable to that of native IgE would result.

That the monomeric polypeptide of the invention has the ability to bindto the high-affinity receptors of mast cells and basophils is quitesurprising. Firstly, the fact that the chain is monomeric at all isunexpected since it would have been anticipated that after synthesis ofthe peptide chain there would be an immediate spontaneous dimerisationof peptide chains via their cysteines at location 318 (28 in FIG. 2).The cysteines at locations 231 and 318 have been previously implicatedin the formation of the inter-epsilon chain disulphide bonding in IgE.The surprising existence of the monomeric polypeptide of this inventionwhich includes the cysteine at location 318 suggests that oneexplanation of this unexpected occurrence is that the inter epsilonchain disulphide pairing in IgE is not, as previously believed, of thehomotypic (AA,BB) type but of the heterotypic (2AB) type. Secondly, thebinding activity of the monomeric polypeptide is quite unexpected sinceit has previously been believed that two epsilon chains were necessaryfor the binding at mast cell receptor sites to trigger exocytosis. Inthe same study, loss of binding activity was found to occur when theinter epsilon chain bonding at location 318 was broken even although thechains remained covalently linked via the more resistant disulphideinterchain bond assumed to link the two cysteines at location 241. Thepolypeptide of the invention lacks sequences necessary for the formationof any of the three immunoglobulin domains which exist in human IgE andthus this invention indicates that this dimeric structural framework isnot essential in its entirety for recognition by the high affinityreceptors of the mast cells.

The core sequence of the invention is less than a quarter of the size ofthe Fc region of the IgE heavy chain, having a molecular weight ofaround 10,000. The amino acid sequence of the polypeptide spans theC-terminal end of the CH2 domain and the N-terminal end of the CH3domain and incorporates a beta-turn from each of the two domains and theso-called "hinge" between them.

A method for producing the polypeptide utilising genetically modifiedEscherichia coli containing a DNA insert coding for the core amino acidsequence will now be described in the following Example 1.

EXAMPLE 1

Human myeloma cell line 266B1 which had previously been used by Bennich(Prog. in Immunol. Vol I, North Holland Publishing Company, pages 49 to58, 1974) contains a functional epsilon gene sequence that can easily becloned.

The known amino acid sequence of IgE provided all the informationrequired to make an oligonucleotide probe for screening a cDNA libraryfrom the 266B1 cell line. The original cell line synthesised from 2 to 7micrograms of IgE per 10⁶ cells per 48 hours. During propagation of theline and adaptation to growth in suspension culture, the synthesis ofIgE had evidently declined, the levels obtained being about 20 nanogramsof IgE per 10⁶ cells per 48 hours. The synthesis of IgE was confirmed bylabelling the protein in culture and SDS polyacrylamide gelelectrophoresis of the fraction precipititated from the culturesupernatants by anti-human IgE Fc anti-serum. Similar analysis of thefraction precipitated by anti- human lambda light chain antiserumdemonstrated the presence of a twenty-fold excess of monomeric overIgE-associated lambda light chain in the secreted immunoglobulin.Despite the poor expression of the epsilon gene in 266B1, the level ofmRNA was sufficient for the task of cDNA synthesis and cloning.

Total RNA was extracted from 266B1 cells and mRNA was purified byoligo-dT chromatography. The presence of intact epsilon chain mRNA wasdemonstrated by translation into polypeptide chains immunoprecipitableby goat anti-human IgE and having the expected electrophoretic mobilityin SDS polyacrylamide gels corresponding to the 66,000 daltonunglycosylated human epsilon chain. The epsilon chain mRNA was enrichedby a factor of ten by sucrose gradient centrifugation, the relativeconcentrations of epsilon chain mRNA in the different fractions beingmonitored both by the translation assay and by oligonucleotide-primedsynthesis of cDNA of the expected length.

Double-stranded cDNA was enzymatically synthesised using routineprocedures and the cDNA was recombined by means of linkers into aappropriate restriction site in a plasmid vector and transformed into E.coli.

An oligonucleotide probe of eleven nucleotides was designed on the basisof the amino acid sequence of the protein previously determined byconventional amino acid sequencing techniques and was chemicallysynthesised. The probe itself failed to detect any cDNA clones butserved as a satisfactory primer for cDNA synthesis, permitting theacquisition of additional sequence information by DNA sequencing. A new22 nucleotide-long probe was constructed, based on this sequence and thelarger probe detected five positive cDNA clones out of a total of 500.The cDNA inserts of the positive clones were excised by digestion withthe appropriate restriction endonuclease and the sizes were found to bein the range of from 0.6 to 2.0 kb; only the largest, 2 kb clone,designated pJJ71, was extensively characterised. It contains thesequences correponding to the 5' and 3' untranslated regions of themRNA, plus those encoding the amino terminal secretion peptide of twentyamino acids and the entire mature epsilon chain.

Reference is now made to the accompanying drawing which shows thederivation of the plasmid pE2-3, the expression plasmid directing thesynthesis of the polypeptide of the invention. The human epsilon DNAcoding sequence is represented by the box V indicating the variableregion, and C1 to C4, the four constant domains. The solid arrows denotethe inducible promoters mediating transcription of sequences cloneddownstream. In ptac-85 and its derivative pE49 the tac promoter ispresent: the lambda P1 promoter is used by vector pAS1 and recombinantspASE1 and pE2-3. The synthetic DNA translation terminator in pE2-3 hasthe sequence 5'-GCTTAATTAATTAAGC-3'.

Expression of the polypeptide in E. coli was achieved via threesubclonings of epsilon Fc cDNA cloned in pJJ71. First, the Sa1I-PvuIIfragment corresponding to epsilon Fc and some forty base pairs ofuntranslated sequences, after digestion with S1 nuclease, was ligatedinto the filled NcoI site of ptac-85. The resulting plasmid, pe49,directs the expression of epsilon Fc and introduces a unique Sa1I siteat the 3' end of the truncated flanking sequences. Second, pe49,linearised by SacI and treated with the double stranded exonucleaseBa131, was recleaved by Sa1I and the DNA fragment corresponding to aminoacids 291 to 537 of epsilon Fc was subcloned into pASI. The pASI hadbeen treated with BamHI and Sa1I restriction enzymes (the BamHI sitehaving been made blunt-ended using DNA polymerase) in order to havecompatible termini to those bounding the fragment from pe49. Theresulting plasmid pASe1 directed the synthesis of an epsilon fragmentcomprising the third and fourth domains and part of the second domainfrom amino acid 291. Third, the expression product of pASE1 wasforeshortened at its carboxy terminus by introducing a translationtermination signal into the cloned DNA at a SmaI site in the positioncorresponding to amino acid 365. The construct, pE2-3, was generated byblunt ligation of a synthetic DNA fragment which contains translationalstop codons in all three reading frames to pASE1 DNA linearised withSmaI.

The polypeptide of the invention was obtained when E. coli strain N4830harbouring pE2-3 was grown under inducing conditions. Expression iscontrolled by the lambda cI repressor which shuts off transcription fromthe lambda PL promoter. E. coli strain N4830 contains a thermolabile cIrepressor which is active at 30 degrees and inactive at 42 degreesCentigrade. A culture of N4830/pE2-3 was thus grown under non-inducingconditions at 30 degrees Centigrade to an A₆₀₀ of 0.8 then heat-shockedat this density by addition of an equal volume of medium preheated to 65degrees Centigrade. After repressor inactivation the culture was grownat 42 degrees Centigrade for a further three hours and then harvested.Electrophoresis of a lysate of this culture showed the presence of a 10Kpeptide (not present in the absence of induction) visible on Coomassiestaining, and shown immunologically to be an epsilon derivative byWestern blotting. The expected size of the product of the gene fragmentis 9,500 daltons.

The polypeptide was present in the lysate as insoluble material whichwas recovered by dissolution in 8M urea. The peptide remained solubleafter removal of urea by dialysis and was purified to near homogeneityby anti-human epsilon affinity chromatography. Polyacrylamide gelelectrophoresis under non-reducing (as well as reducing) conditionsshowed that the purified polypeptide had a molecular weight of about10,000, indicating that unreduced peptide was monomeric.

EXAMPLE 2

The effectiveness of the polypeptide of the invention was compared withnatural IgE and various fragments thereof in a series of tests using thepassive cutaneous anaphylaxis (PCA) reaction [described in Nature 315:577-578 (1985)]. The results are presented below in Table I.

                  TABLE I    ______________________________________                  Heavy-chain           Amino  Domains            Ac-    Source   acids    VH     CH1  CH2  CH3  CH4  tivity    ______________________________________    Myeloma IgE              1-457   +      +    +    +    +    +    (PS)    pSC213   208-537  -      -    +    +    +    +    pES1     300-537  -      -    p    +    +    +    PE delta 4             209-429  -      -    +    +    -    ±    pE2-3    291-366* -      -    p    p    -    +    ______________________________________     p = part of domain     *amino acid sequence 1 to 76 shown in FIG. 2

The approach to intervention in the allergic response adopted in thepresent invention is to block IgE high affinity receptor sites byadministration to the patient of an amount of the polypeptide of theinvention. This approach is believed to leave the low-affinity receptorsunaffected and free to participate in their apparent immunological role.

From the results summarised in Table I, it can be seen that a positiveeffect is attained with all the sequences quoted, thus narrowing downthe binding sites of IgE to mast cells to the seventy-six amino acidsequence of this invention. This sequence displayed an affinity constantfor the human basophil receptor (5 ×10⁹ /mol) which wasindistinguishable from that of a myeloma IgE.

Inhibition of the Prausnitz-Kustner reaction was also displayed by thefragments listed in Table 1 above, that is, by amino acid sequenceswhich contain the sequence 1 to 76 shown in FIG. 2, but, no inhibitionwas found for three other fragments of IgE, namely:

(i) amino acids spanning locations 430 to 537 of the IgE sequence, andtherefore containing no residues in common with the polypeptide of theinvention,

(ii) amino acids spanning residues 208 to 326, and therefore containingthe residues 1 to 35 of the polypeptide of this invention; and,

(iii) amino acids spanning residues 329 to 537, and therefore containingthe residues 38 to 76 of the polypeptide of this invention.

The results of the P-K reaction tests were as follows:

Inhibition of the P-K Reaction by IgE Fragments

A single subject was used for passive sensitisation. The serum IgE ofthis subject was 4 IU/ml (approximately 10 ng/ml). The sensitising serum(E.C.) contained 380 IU/ml of IgE (912 ng/ml serum E.C.), of which 8.7%was directed against ragweed antigen, as determined by the specific dropin serum IgE following absorption of the serum over a Sepharose 4Bragweed antigen column compared with a control Sepharose 4B human serumalbumin. Serum E.C. was free of detectable hepatitis B antigen and ofantibodies to that antigen and to human immunodeficiency virus (HIV).Serum E.C. was obtained in 1983 and its donor is currently (1987)healthy and HIV antibody negative. Epsilon chaim fragments were injectedinto skin sites one hour before the injection of serum E.C. Skin siteswere challenged 48 hours later with ragweed antigen (1,000 proteinnitrogen units/ml of a mixture of giant and short ragweed). Twentyminutes later the skin sites were examined for the presence of wheal anderythema. The surface area of the reaction was estimated as follows:Transparent tape was used to transfer the outline for the reactions topaper which was then cut out and weighed on an analytical balance. Thearea was read from a standard curve. All injections were intradermal and0.02 ml in volume. In each experiment a set of skin sites was alsosensitised with diluent. None of these sites showed wheal or flare whenchallenged with ragweed antigen. The diluent consisted of 0.15M sodiumchloride and 0.03% human serum albumin. In both experiments, reported inTable 2 below, skin sites were sensitised with a 1:100 dilution of serumE.C. containing 5×10⁻¹¹ M IgE.

                  TABLE 2    ______________________________________                  Area of Wheal & Flare                    Expt. 1  Expt. 2    Inhibitor       10 ug/ml 1 ug/ml    ______________________________________    Diluent          65/380   92/455    IgE (P.S.)      0/0      0/0    aa 208-537      0/0      0/0    aa 291-537      0/0      0/0    aa 209-429      0/0      0/0    aa 291-366*     0/0      0/0    aa 430-537       60/416   85/438    aa 208-326       70/350   80/405    aa 329-537       69/375   78/398    ______________________________________     * = aa 1 to 76 in FIG. 2.

Relative Activity of Recombinant IgE (ND) Peptides in the Inhibition ofthe P-K Reaction

The molarities of the epsilon chain fragments were calculated takinginto account the proportion of dimers versus monomers in eachpreparation. Monomers were included in the calculations because thepolypeptide of the invention has never existed in detectable dimeric oroligomeric forms and it was a potent inhibitor of the P-K reaction (seeTable 2 above). Each fragment was used over a range of 10⁻¹³ to 10⁻⁶ Min ten-fold increments. The results are presented in Table 3 below.

                  TABLE 3    ______________________________________    Molarity required for 50% inhibition    of the P-K reaction reduced by    Expt 1              Expt 2    Serum E.E. dil 1:100                        Serum E.C. dil 1:20    = 5 × 10.sup.-11 M IgE                        = 2.5 × 10.sup.-10 M IgE    Source  Molarity  % Potency Molarity                                        % Potency    ______________________________________    IgE (P.S.)            2 × 10.sup.-10                      100       2 × 10.sup.-9                                        100    aa 208-537            4 × 10.sup.-10                      50        4 × 10.sup.-9                                        50    aa 291-537            5 × 10.sup.-10                      40        4 × 10.sup.-9                                        50    aa 209-429            5 × 10.sup.-10                      40        6 × 10.sup.-9                                        33    aa 291-366*            6 × 10.sup.-10                      33        5 × 10.sup.-9                                        40    ______________________________________     * = 1 to 76 in FIG. 2.

Duration of the Inhibition of the P-K Reaction

In Table 4 below, values are given in days elapsed following theinjection of the inhibitor before a successful P-K reaction could beachieved. Multiple skin sites of a normal subject were injected at day 0with IgE (P.S.) the polypeptide of the invention or diluent. Atintervals (days 0,4,9,12,14,17,19,21) individual skin sites weresensitised with a 1:100 dilution of serum E.C. (5×10¹¹ M IgE) thenchallenged 48 hours later with ragweed antigen. Sites pretreated withdiluent always gave a positive wheal and flare reaction with a meanstandard deviation of the flare of 392±58 mm for the eight successivedeterminations. The days shown in Table 4 represent the time of thefirst appearance of flare and/or erythema at the challenged skin sites.

                  TABLE 4    ______________________________________                  Inhibitor Concentration    Inhibitor       10.sup.-7 M                            10.sup.-6 M    ______________________________________    IgE (P.S.)      12      19    aa 301-376*      9      14    ______________________________________     *= 1 to 76 in FIG. 2.

We claim:
 1. A polypeptide which is capable of binding specifically tothe high affinity Ec receptor sites for IgE which exists on human cellsand which has the following amino acidsequence:Gln-Lys-His-Trp-Leu-Ser-Asp-Arg-Thr-Tyr-Thr-Cys-Gln-Val-Thr-Tyr-Gln-Gly-His-Thr-Pha-Glu-Asp-Ser-Thr-Lys-Lys-Cys-Ala-Asp-Ser-Asn-Pro-Arg-Gly-Val-Ser-Ala-Tyr-Leu-Ser-Arg-Pro-Ser-Pro-Phe-Asp-Leu-Phe-Ila-Arg-Lys-Ser-Pro-Thr-Ile-Thr-Cys-Leu-Val-Val-Asp-Leu-Ala-Pro-Ser-Lys-Gly-Thr-Val- Asn-Leu-Thr-Trp-Ser-Arg.
 2. Apolypeptide which is capable of binding specifically to the highaffinity Fc receptor sites for IgE which exists on human cells and whichhas the following amino acidsequence:X-Gln-Lys-His-Trp-Leu-Ser-Asp-Arg-Thr-Tyr-Thr-Cys-Gln-Val-Thr-Tyr-Gln-Gly-His-Thr-Phe-Glu-Asp-Ser-Thr-Lys-Lys-Cys-Ala-Asp-Ser-Asn-Pro-Arg-Gly-Val-Ser-Ala-Tyr-Leu-Ser-Arg-Pro-Ser-Pro-Phe-Asp-Leu-Phe-Ile-Arg-Lys-Ser-Pro-Thr-Ile-Thr-Cys-Leu-Val-Val-Asp-Leu-Ala-Pro-Ser-Lys-Gly-Thr-Val- Asn-Leu-Thr-Trp-Ser-Arg- Y,inwhich X and Y are oligopeptide sequences initiating and terminating thechain which do not prevent said polypeptide from binding specifically tothe high affinity Fc receptor sites on human cells and are notphysiologically harmful.
 3. A method of preparing the polypeptideclaimed in claim 2, said method comprising the steps of:culturing a hosttransformed by a vector comprising a DNA segment having the followingnucletide sequence: CAG AAG CAC TGG CTG TCA GAC CGC ACC TAC ACC TGC CAGGTC ACC TAT CAA GGT CAC ACC TTT GAG GAC AGC ACC AAG AAG TGT GCA GAT TCCAAC CCG AGA GGG GTC AGC GCC TAC CTA AGC CGG CCC AGC CCG TTC GAC CTG TTCATC CGC AAG TCG CCC ACG ATC ACC TGT CTG GTC GTC GAC CTG GCA CCC ACC AAGGGG ACC GTG AAC CTG ACC TGG TCC CGG said segment being oriented withinsaid vector such that in a host said segment is expressed to produce apolypeptide; and isolating the polypeptide from the culture.
 4. A methodof preparing the polypeptide, said method comprising the stepsof:culturing a host transformed by a vector comprising a DNA segmenthaving the following nucleotide sequence: CAG AAG CAC TGG CTG TCA GACCGC ACC TAC ACC TGC CAG GTC ACC TAT CAA GGT CAC ACC TTT GAG GAC AGC ACCAAG AAG TGT GCA GAT TCC AAC CCG AGA GGG GTC AGC GCC TAC CTA AGC CGG CCCAGC CCG TTC GAC CTG TTC ATC CGC AAG TCG CCC ACG ATC ACC TGT CTG GTC GTCGAC CTG GCA CCC ACC AAG GGG ACC GTG AAC CTG ACC TGG TCC CGG isolatingthe polypeptide from the culture to obtain a polypeptide as claimed inclaim 17; and treating that polypeptide to remove the chain initiatingand terminating groups X and Y.
 5. In a conpetitive binding assay inwhich a competitor is used, the improvement being said competition isthe polypeptide claimed in claim
 1. 6. In a competitive binding assay inwhich a competitior is used, the improvement being said competitor isthe polypeptide claimed in claim
 2. 7. A diagnostic kit including meansfor conducting a competitive binding assay and including, for use as acompetitor in said assay, a polypeptide as claimed in claim
 1. 8. Adiagnostic kit including means for conducting a competitive bindingassay and including, for use as a competitor in said assay, apolypeptide as claimed in claim 2.